Assays and methods for diagnosing substance use disorder

ABSTRACT

Assays and methods for verifying the validity of a urine sample submitted for Drugs of Abuse (DOA) testing. Embodiments include a SUD Diagnostic Panel that includes six assays: specific gravity index assay, long-duration counterfeit urine assay, short-duration counterfeit urine assay, oxidant history assay, pH assay, and creatinine assay. The SUD Diagnostic Panel detects twelve principle classes of adulteration. Detection of adulteration of one or more urine samples from a patient indicates an attempt to subvert test results and provides an objective indication in one instance and an object diagnosis in another instance of SUD.

BACKGROUND OF INVENTION

Substance use disorder (SUD) is a condition in which a patient exhibitsa pathologic pattern of behavior of continued use of a substance despiteexperiencing significant negative consequences related to such use.Manifestations of SUD include impaired control of use, socialimpairment, risky use behavior, and efforts to hide use. There may alsobe physiological manifestations such as changes in brain function.

The substance drugs of abuse (DOA) utilized by those suffering with SUDare typically those that are subject to subversion techniques. Teenagechildren are a particularly vulnerable class. Overdose deaths in thisclass exceed deaths from school shootings and other cases of deathcombined. Unfortunately, parents and teachers have only behavioralobservations to question if an individual has SUD. But, these sameobservations could result from other psychiatric or behavioral problems.The internet and other social media provide a plethora of informationabout how to subvert or “beat” a drug test and subversion products canbe easily obtained. For this reason, neither parents nor teachers orphysicians cannot rely upon current drug screening for diagnosis. Worse,negative drug results lead to the belief that symptoms are caused byother factors and treatment is not undertaken. This problem is notlimited to teenagers. The overall detection of DOA has been seriouslycompromised due to the wide use of subversion agents and the success ofthese agents in masking detection. Thus, the metric data relied on todetermine the targeting means used to combat the current opioid crisisis seriously flawed. These flaws in the data that is relied on degradethe effectiveness of providing treatment and recovery services andcorrespondingly adds greatly to the national costs related to drug useand abuse.

There are currently twelve recognized principle classes of subversion,which are used to adulterate or substitute for a patient's urine sample:(1) Alteration of pH, (2) simple dilution either in vivo or in vitro,(3) in vivo dilution with creatine/protein/water loading, (4) salting(e.g., baking soda), (5) oxidant adulteration (e.g., Stealth™), (6)gluteraldehyde adulteration, (7) heavy metal adulteration, (e.g. zinc)(8) substitution with synthetic (Counterfeit) urine, (9) substitute withurine from another person, (10) sulfhydryl blocking agents, (e.g.,iodoacetamide) (11) cationic detergents, (e.g., dimethylbenzylalconiumchloride), and (12) proteases, (e.g., bromelain).

Laboratories typically screen samples for DOA with automated equipment,such as clinical analyzers, using commercially available EnzymeImmunoassays (EIA) and related methods and reagents. Current screeningmethods utilize bodily fluid or hair to detect the presence of a drug inthe urine and may attempt to assess the validity (absence of subversion)of the sample by measuring or detecting pH, creatinine, reflex specificgravity, and general oxidant use. Nonetheless, the positivity rate forworkplace drug testing has been steadily declining for the last decade.During the same period, deaths from drug overdoses have steadilyincreased. This indicates that the current testing protocols arefailing. Without the ability to test the validity of a sample,proceeding directly to a test for the presence of a drug can result ininaccurate results. For example, it has been shown that the currentassays and methods used for screening samples are effective only fordetecting the first two classes of subversion, which represents lessthan 20% of total subversion efforts and that percentage is declining.

There has been increased evidence and better understanding of thebiological processes that underlie compulsive drug use. This hasresulted in SUD being recognized as a medical illness, opening the doorto numerous forms of treatment that can now be used to help patients.However, as with many diseases, it is important that the diagnosis ofSUD be made early as the disease becomes more difficult to treat as thebrain chemistry of the SUD victim changes. Too often the diagnosis isestablished only after the first overdose. This is the equivalent ofdiagnosing diabetes only after the diabetic suffers a diabetic coma.Combatting the problem must first start with more effective screeningfor drug use so that SUD can be identified.

It is also important that improved assays and methods used for screeningbe useable in automated equipment. This will provide a quicker and morecost effective method for conducting tests, ensuring their continued andfrequent use.

BRIEF SUMMARY

The subject invention successfully addresses the disadvantages with thepreviously known drug use screening methods and provides certainattributes and advantages, which have not been possible by those knownprocedures. In particular, the subject invention provides novel,relatively inexpensive, and highly effective improvements to currentlyknown screening assays. More specifically, the subject inventionprovides a screening panel capable of verifying sample validity bydetecting use of one or more of the twelve recognized classes of druguse subversion techniques. The subject invention can also employ methodsof use that improve the results of DOA testing protocols to moreaccurately diagnose SUD.

The screening panel includes six assays that, when used together, candetect all twelve of the recognized principle classes of subversion. Thescreening assay methods of the subject invention can be employed inadvance of drug screening tests, to verify the validity of the samplebefore additional time and cost is spent conducting further tests. Thescreening method includes three assays that can measure the presence orabsence of specific bio-markers in a urine sample, a specific gravityindex assay that can measure the sodium and potassium in a urine sample,an improved creatinine assay, and an improved pH assay. These combinedassays provide a screening panel and method, referred to herein as the“SUD Diagnostic Panel” the results of which can accurately determine ifa urine sample has been adulterated by any of the twelve known classesof subversion techniques. In order for the SUD Diagnostic Panel toremain effective, it is important that all known means of subversion bedetectable. If, for example, only a Counterfeit urine assay is added dueto its currently common use, the internet and social media will rapidlyreport that drug users are being “busted” for using counterfeit urineand will recommend other subversion means. This will render theCounterfeit urine assay obsolete in the absence of other assaytechniques.

Advantageously, embodiments of the assays used in the SUD DiagnosticPanel are capable of being utilized in standard laboratory automationequipment, such as chemical analyzers typically used to facilitateautomated urine sample analysis. Specifically, spectrophotometry andLC/MS analyses of a sample treated with the assay embodiments of thesubject invention can be used to indicate whether a sample contains orhas contained an adulterant or has otherwise been subjected toadulteration or substitution.

Attempted subversion of a drug test can be a manifestation of SUD andindicates a compelling need to continue use of the substance. Anindication of subversion is thus an objective indication of a behavioraldisorder indicative of SUD. Combating the problem of SUD should firststart with the 6 assay “SUD Diagnostic Panel.”

Specific methods of use of the SUD Diagnostic Panel withspectrophotometric and LC/MS protocols employed for DOA testing canensure that such analyses are conducted on valid samples, therebyreducing unnecessary tests. Furthermore, failing any one of the 6 assaysprovides a provisional diagnosis of SUD. Confirmatory testing is thenmore accurate because passing the SUD panel will allow only validsamples to proceed to drug screening tests. Donors of failed SUD paneltesting should be required to provide a new sample under the most securecollection means. That sample can then proceed to Liquid or Gaschromatography/Mass Spectrophotometry (LC/MS) testing for a broaderrange of drugs than available with EIA screening. Alternatively, theretaken sample can be subjected to the SUD Diagnostic Panel to ensurevalidity prior to proceeding with LC/MS testing.

BRIEF DESCRIPTION OF DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In order that a more precise understanding of the above recitedinvention can be obtained, a more particular description of theinvention briefly described above will be rendered by reference tospecific embodiments thereof that are illustrated in the appendeddrawings. Understanding that these drawings depict only typicalembodiments of the invention and are not therefore to be considered aslimiting in scope, the invention will be described and explained withadditional specificity and detail through the use of the accompanyingdrawings.

FIG. 1A is a flowchart illustrating a procedure by which an embodimentof a SUD Diagnostic Panel, according to the subject invention, can beused to detect an invalid and potentially adulterated urine sample andthe various procedures that can be taken when the sample is determinedto be valid and when the sample is determine to be invalid.

FIG. 1B is a flowchart illustrating an alternative procedure by which anembodiment of a SUD Diagnostic Panel, according to the subjectinvention, can be used to detect an invalid and potentially adulteratedurine sample and used again on a retaken urine sample to detect whetherit is invalid and also potentially adulterated.

FIG. 2 is a graph of an Oxidant History obtained using the Uric AcidEquivalents in a urine sample that has been adulterated with STEALTH, acommercially-available adulterant.

FIG. 3 is a graph of an Oxidant History obtained using the Uric AcidEquivalents in a urine sample that has been adulterated with a solutionof 10% potassium nitrate.

FIG. 4 is a graph of an Oxidant History obtained using the Uric AcidEquivalents in a urine sample that has been adulterated with a solutionof 2% bleach.

FIG. 5 illustrates a method for detecting the presence of alkalinephosphatase (ALP), a long-duration (LD) bio-marker in a urine sample,according to embodiments of the subject invention. The method isillustrated utilizing a dip stick with a Test spot comprisingp-nitrophenyl phosphate substrate to which a sample can be added andthat, according to embodiments of the subject invention, reacts withalkaline phosphatase (ALP) in true urine to form the chromogenp-nitrophenol. The control has neither the substrate nor the chromogen.The p-nitrophenol turns yellow at the alkaline pH necessary for the ALP,if present in the sample, to catalyze the p-nitrophenyl phosphatesubstrate. When an alkaline pH reagent is added to the Control spot, thesample can be counterfeit urine if it also turns dark yellow indicatingthat p-nitrophenol was present in the sample and was not formed as aresult of alkaline phosphatase in the urine. It can also be seen in thisFigure that if the thymolphthalein chromogen was added to the sample,the Test spot, as well as the Control spot turn blue, due to thealkalinity of both spots. Further, if both chromogens were added to thesample, the Test spot and the Control spot turn green, a result of thecombination of both chromogen colors.

FIG. 6 illustrates a method for detecting the presence of acidphosphatase (AP), a short-duration (SD) bio-marker in a urine sample,according to embodiments of the subject invention. The method isillustrated utilizing a dip stick with a Test spot comprising athymolphthalein monophosphate substrate to which the sample can be addedand that, according to embodiments of the subject invention, react withacid phosphatase (AP) present in true urine to form the chromogenthymolphthalein. Thymolphthalein is colorless at the acid pH necessaryfor the AP, if present in the sample, to catalyze the thymolphthaleinmonophosphate substrate. The Control spot has neither the substrate, northe chromogen. When an alkaline pH reagent is added to the Test spot andthe Control spot, the chromogen formed at the Test spot is activated andimparts a blue color to the Test spot. The Control spot can indicatewhether the sample is counterfeit urine if it also turns blue,indicating that thymolphthalein was present in the sample and was notformed as a result of acid phosphatase in the urine. It can also be seenin this Figure that if the p-nitrophenol chromogen was added to thesample, the Test spot, as well as the Control spot turn yellow. Further,if both chromogens were added to the sample, the Test spot and theControl spot turn green, a combination of both chromogen colors.

FIG. 7 illustrates a method for detecting the presence of both acidphosphatase (AP) and alkaline phosphatase (ALP) in a sample, accordingto embodiments of the subject invention. The method is illustratedutilizing a dip stick with one spot comprising p-nitrophenyl phosphatesubstrate (labeled as ALP) and another spot comprising thymolphthaleinmonophosphate substrate (labeled as AP), as well as a third Control spotcontaining neither of the substrates, nor their chromogens. According toembodiments of the subject invention, when true urine is added to theTest spots, the substrates react with either the alkaline phosphatase(ALP) or acid phosphatase (AP) to form the respective chromogensp-nitrophenol and thymolphthalein. If the sample is true urine, the ALPTest spot turns yellow and the AP Test spot and Control spot remaincolorless. When an alkaline pH reagent is added to the AP Test spot andthe Control spot, the AP Test spot turns blue and the Control spotremains uncolored. The Control spot indicates a counterfeit, thusinvalid, sample if it turns either blue or yellow, indicating that thechromogen was likely added to the sample and was not formed as a resultof alkaline or acid phosphatase in the urine. If the control turnsgreen, it indicates that both chromogens were added to the sample andwere not formed as a result of alkaline or acid phosphatase in theurine, thus, an invalid sample. It can also be seen that if eitherchromogen was added to the sample, all of the Test spots, as well as theControl spot turn yellow and/or blue. Further, if both chromogens wereadded to the sample, all of the Test spots and the Control spot turngreen, a combination of both chromogen colors.

FIG. 8 is an equation showing one embodiment of the reaction method ofthe subject invention.

FIG. 9 is a table showing the results of the Specific Gravity Index(SGI) measurements on normal urine samples and samples adulterated withcommon adulterants. Typically, the effects of an adulterant are notmeasureable below 10% w/v. It can be seen that, with embodiments of thesubject invention, adulterant concentrations in a sample of 1% and 5%w/v can be detected and indicate the sample is positive foradulteration. This indicates a high sensitivity for this test.

FIG. 10 is a graph comparing the actual specific gravity of urinesamples to the Specific Gravity Index of the subject invention. Theregression analysis statistics are also provided.

FIG. 11 shows the pH ranges and associated colors for the pH indicatordyes utilized with embodiments of the pH assay of the subject invention.

FIG. 12 is a graph related to the pH assay illustrating that a highabsorbance at the primary wavelength and little absorbance at thesecondary wavelength produces a high positive net absorbance.

DETAILED DISCLOSURE

The subject invention pertains to an objective method of diagnosingSubstance Abuse Disorder (SUD) utilizing an assay panel. Morespecifically, the subject invention provides one or more embodiments ofa screening panel comprising six separate assays capable of detectingwhether a urine sample has been adulterated or substituted by any of thetwelve principle classes of subversion techniques. It has been shownthat the use of subversion techniques is a manifestation of SUD. Thus,detection of subversive behavior can be indicative of SUD.

The following description will disclose that the subject invention isparticularly useful in the field of urine analysis utilizing automatedequipment, such as, chemical analyzers, to detect adulteration orsubstitution of a sample and use of Drugs of Abuse (DOA). A person withskill in the art will be able to recognize other uses that would beapplicable to the devices and methods of the subject invention. Forexample, certain diseases or disease states may also be detectable withthe embodiments of the subject invention. While the subject applicationdescribes, and many of the terms herein relate to, a use for detectingor diagnosing SUD, which is related to use of DOA, other uses andmodifications apparent to a person with skill in the art and havingbenefit of the subject disclosure are contemplated to be within thescope of the present invention.

When the term “about” is used herein, in conjunction with a numericalvalue, it is understood that the value can be in a range of 95% of thevalue to 105% of the value, i.e. the value can be +/−5% of the statedvalue. For example, a temperature of 100° C. means from about 95° C. toabout 105° C.

Substance Use Disorder (SUD) is a recognized mental disorder thatmanifests as behavioral characteristics related to use of Drugs of Abuse(DOA). More specifically, it has been shown that efforts to mask, hide,or otherwise subvert detection of use of DOA is indicative of SUD. Infact, the Substance Abuse and Mental Health Services Administration(SAMHSA) recognizes that behavior changes are a reason to suspect SUD.The ability to detect subversion of test results, usually by one of thetwelve principle classes of subversion, can be an objective indicationof a behavioral change indicative of SUD.

Urine sample analysis with automated equipment using spectrophotometricand LC/MS techniques is the most cost effective means for detecting DOAor their metabolites. The current assay panel used to analyze a urinesample relies on detecting the presence of a DOA in a urine sample. Thecurrent assays used for detecting invalidity of samples for DOA testinginclude analysis of pH, creatinine levels, specific gravity, and generaloxidant detection. These assays are usually capable of effectivelydetecting only two of the twelve classes of subversion—adulteration bymodifying pH and simple dilution. Thus, if any other subversiontechnique is used, the current assay panel can return a false negativeresult for DOA.

Embodiments of the subject invention provide an advantageous improvementto the current assay panel by replacing the current specific gravity andgeneral oxidant assays with a new and more broadly effective SpecificGravity Index assay and an Oxidant History assay. Added to the currentassay panel are two unique, highly effective Long Duration (LD) andShort Duration (SD) assays for detecting Counterfeit Urine. Alsodisclosed herein are improved pH and creatinine tests. The SUDDiagnostic Panel embodiments of the subject invention provide six assayscapable of detecting all twelve of the recognized subversion techniques:Counterfeit (1) Alteration of pH, (2) simple dilution either in vivo orin vitro, (3) in vivo dilution with creatine/protein/water loading, (4)salting (e.g., baking soda), (5) oxidant adulteration (e.g., Stealth™),(6) gluteraldehyde adulteration, (7) heavy metal adulteration, (e.g.,zinc), (8) substitution with synthetic (Counterfeit) urine (9)substitution with urine from another person, (10) sulfhydryl blockingagents, (e.g., iodoacetamide) (11) cationic detergents, (e.g.,dimethylbenzylalconium chloride), and (12) proteases, (e.g., bromelain).Alternative embodiments can include fewer than the six assays.Utilization of the full six assay screening panel can, however, providethe most reliable objective diagnosis of SUD.

The following Table 1 lists the 12 currently known methods ofadulteration, the assay useful in detecting such adulteration, and theconditions under which adulteration is confirmed, as described herein:

TABLE 1 Subversion Techniques and methods of detection Method ofAdulteration or Criteria for Substitution: Assay for Detection:Confirmation: Change of pH pH Assay pH less than 3 and greater than 11Simple dilution, Specific Gravity Index (SGI) Creatinine < 20.0 in vivoor in Assay and Creatinine Assay and/or SGI < 1.0030 vitro In vivodilution Specific Gravity Index (SGI) SGI < 1.0030 with creatine/ Assayprotein/water loading Salting Specific Gravity Index (SGI) SGI > 1.0350Assay Oxidant Oxidant History Assay Uric Acid Equivalents < 10Gluteraldehyde Specific Gravity Index (SGI) SGI < 1.0030 and Assay andLong-Duration (LD) LD < 8 Assay Heavy Metal Specific Gravity Index (SGI)SGI < 1.0030 Assay Substitution Long-Duration (LD) Assay LD: Females <pH 9; with synthetic and Short-Duration (SD) Males < pH 12 (Counterfeit)Assay SD: Females < pH 9 urine product Males < pH 12 SubstitutionShort-Duration (LD) Assay SD: Females < pH 9; with the urine Males < pH12 of another person Sulfhydryl Specific Gravity Index (SGI) SGI <1.0030 blocking Assay agents cationic Specific Gravity Index (SGI) SGI <1.0030 detergents Assay Protease Specific Gravity Index (SGI) SGI <1.0030 Assay

As shown in FIG. 1, embodiments of a SUD Diagnostic Panel can beincorporated into the current methods for analyzing a urine sample afterfirst testing whether a sample is valid, or if it has been subjected toadulteration or substitution. A SUD Diagnostic Panel can be conductedprior to any tests conducted for DOA or metabolites thereof. If the SUDDiagnostic Panel indicates that the sample is valid, further testing bystandard Enzyme Immunoassays (EIA) can be conducted and, because theurine sample was validated, the results can be considered accurate.

As discussed above, SUD is characterized by the patient taking steps tohide their use of DOA. Their reliance, a.k.a. addiction, to the DOAcauses them to engage in risky behaviors, such as attempts to subvertDOA tests. In one embodiment, if the SUD Diagnostic Panel indicates thatthe sample is invalid, results can be an initial indication of SUD. Atthat point, steps can be taken to secure another sample under closerscrutiny.

In one embodiment, the retaken sample can be retested with the SUDDiagnostic panel to again determine if the retaken sample is valid orinvalid. If the retaken sample is determined to be invalid, furthertesting can be avoided and an appropriate report provided. In oneembodiment, an invalid retaken sample can be considered furtherobjective indication of SUD. The use of adulteration techniques tosubvert DOA tests can be considered a risky behavior. The reliability ofthe SUD Diagnostic Panel in detecting adulteration of urine samples canmake it an important step in detecting such risky behavior. In analternative embodiment, a SUD Diagnostic panel result that indicates aninvalid retaken sample can be considered an objective diagnosis of SUD.

If the retaken test is determined to be valid, additional and more exactLiquid or Gas Chromatography/Mass Spectrophotometry (LC/MS) tests can beconducted to determine the presence of DOA. In one embodiment, adiagnosis of SUD can be objectively confirmed if the LC/MS testsindicate DOA in the retaken sample.

Unlike the enzyme immunoassay procedures (EIA), LC/MS procedures candetect a much wider spectrum of DOA. Thus, confirmation of DOA in asample using LC/MS can be an important step in the objective diagnosisof SUD. Many drugs are present in urine samples as glucuronidemetabolites. It is known that detection of these glucuronide metabolitescan be inexact and inaccurate with LC/MS procedures for several reasons.For the LC/MS procedure to be effective, the enzyme glucuronidase isused to hydrolyze the glucuronide metabolites to create a free form of adrug that may be present in the urine sample, which can then be detectedby LC/MS procedures.

The inventor has discovered that the effectiveness of LC/MS procedurescan be reduced when the sample is subjected to certain classes ofsubversion techniques listed in Table 1. Specifically, the glucuronidaseenzyme activity is destroyed by heavy metal adulteration, such as withp-chloromercuriobenzoate (PCMB) and also with sulfhydral blockers, suchas iodoacetamide (IAA). The use of these types of adulterants and theirinactivation of the glucuronidase enzyme can adversely affect LC/MSprocedures and results and reduce its effectiveness in confirming adiagnosis of SUD.

The SUD Diagnostic Panel provides the advantageous ability to detectadulterants that can affect LC/MS procedures and results. FIG. 2illustrates an example of a method for analysis of samples utilizing theSUD Diagnostic panel. In one embodiment, the SUD Diagnostic Panel isutilized to analyze a urine sample to determine validity. In a furtherembodiment, the SUD panel is utilized on retaken samples beforeconducting LC/MS DOA testing procedures. This can provide LC/MS resultsthat can be relied on to diagnose SUD.

Utilization of the full SUD Diagnostic Panel, with all six assays, canmost accurately indicate the use a subversion technique. The methods ofusing the SUD Diagnostic Panel with both initial and retaken urinesamples can provide an effective means for objectively diagnosing SUD.Each of the assays in the SUD Diagnostic panel and their mode ofoperation are discussed in detail below.

I. Oxidant History Assay Utilizing Bio-Markers in Urine

Embodiments of an Oxidant History assay can be used alone or to createan Oxidant History (OHist), which utilizes two or more marker values todemonstrate historical changes in the sample markers during apre-determined time window. U.S. Pat. No. 10,082,495 describes anOxidant History Assay that can be utilized with embodiments of thescreening panel of the subject invention. The entirety of U.S. Pat. No.10,082,495 is hereby incorporated by reference.

Advantageously, the results obtained with an OHist can be unaffected bythe timing of the tests and are effective when utilized with automatedlaboratory equipment. The initial physical change and concentration ofthe markers caused by the presence of an oxidant-adulterant can remainstable over time. Any change in the concentration of the markers canreinforce or confirm the presence of an oxidant and, thus, adulterationof the urine sample. Advantageously, the marker(s) utilized arenaturally present in urine of all primates, including humans.

In one embodiment, uric acid is utilized as a marker in a urine sample.A phosphotungstate reagent can be used to detect the amount of uric acidin the sample. However, phosphotungstate lacks specificity for detectionof uric acid. This is believed to be due to the presence of low levelsof secondary compounds or constituents, which can be referred to as“non-urate markers” that are found in urine. These non-urate markersinclude, but are not limited to, ascorbic acid, cystine, cysteine,ergothioneine, and glutathione. These non-urate markers, referred toherein as a Uric Acid Equivalent markers were discovered to also beoxidized when exposed to oxidizing adulterants. An OHist assay of thesubject invention advantageously employs, or is at least not inhibitedby, this lack of specificity of phosphotungstate and can use the UricAcid Equivalent beneficially for the indirect detection ofoxidizing-adulterants in urine. Furthermore, the presence of uric acidand other non-urate markers in urine allow phosphotungstate to be usedas a chromogen. Specifically, the phosphotungstate reagent of the OHistassay can react with the Uric Acid Equivalents causing urine to turn ablue color, thus enabling quantification of these substances. In thepresence of an oxidative-adulterant, the Uric Acid Equivalents can bereduced, lightening the blue color formation and, at certainconcentrations, an oxidative-adulterant can eliminate most or all of theblue color. This can result in a reduction in light absorbance atcertain wavelengths during colorimetric or spectrophotometric analysis.Advantageously, the embodiments of the subject invention can be usedwith colorimetric techniques and spectrophotometric devices that canmore accurately and easily quantify the amount of Uric Acid Equivalentsin the sample of bodily fluid, such as, for example, urine. The additionof oxidative-adulterants, which normally inhibit drug measurement inurine by oxidatively destroying the drugs, can also oxidize uric acidand non-urate markers. The oxidation of these markers can significantlyaffect their ability to react and form a blue color reaction with thephosphotungstate reagent of the present invention, which is easilydetectable by spectrophotometric techniques.

The blue coloration, when analyzed spectroscopically, can indicate theconcentration of markers present in the urine sample. In one embodiment,the blue coloration affects light absorbance in the range of frombetween approximately 580 to approximately 800 nm. In a more particularembodiment, the blue coloration affects light absorbance in the range offrom between approximately 600 to approximately 700 nm. In a specificembodiment, the blue coloration affects light absorbance in the range offrom between approximately 650 nm to approximately 700 nm.

Ideally, a urine sample with a Uric Acid Equivalent or OHist panelresult that the laboratory considers borderline or otherwise suspiciousshould be retested 24 hours after collection. The oxidant time-historyof a sample, as provided by the OHist testing procedures of the subjectinvention, can provide definitive confirmation of oxidant adulterationof a urine sample. An OHist, as used herein, refers to obtaininghistorical light absorbance measurements of Uric Acid Equivalents valuesin a sample over a pre-determined period of time or during a timewindow. These light absorbance measurements can be used to prepare anOHist for the given time window, which can be a history of the effectsof an oxidative-adulterant on a urine sample, if present. As discussedabove, the OHist can show how an oxidant adulterant in a urine sampleprogressively destroys the phosphotungstate-reducing ability of themarkers in the urine sample, thereby affecting the light absorbance ofthe sample at certain wavelengths. Although the initial decrease can besubstantial, as indicated by the graphs in FIGS. 2, 3, and 4, theeffects can continue, often more slowly, over time.

The level of Uric Acid Equivalents can vary between samples. However,the embodiments of the subject invention can still be effective becausethe effects of the oxidative-adulterants can continue to reduce the bluecolor in a sample to levels that are far lower than the lowest end ofthe range possible. A low cut-off limit can be established to inhibitfalse positive results, but which can also inhibit false negativeresults. However, with the embodiments of the subject invention, anoxidative-adulterant that can affect measurements of samples treatedwith the phosphotungstate reagent of the subject invention can be moreeasily detected by confirmation testing when conducted at a later time.Absent the presence of an oxidative-adulterant, the levels of uric acidin urine are known to be very stable for a relatively long time period,at least several days. The confirmation test can be carried out byre-assaying and observing changes in the level of reduction capacity ofthe phosphotungstate reagent, which can only be caused by the presencein the sample of an oxidative-adulterant affecting the markers that makeup the Uric Acid Equivalent value. A significant lowering of thephosphotungstate reduction capacity of the urine sample, as indicated bya reduction in the blue coloration of the urine, can thus be adefinitive confirmation of the presence of oxidative adulterant,regardless of the original uric acid concentration. Thus, themeasurement of phosphotungstate reduction or the levels of the markers,and changes therein, can be useful in both a screening test and aconfirmation test.

Certain embodiments of the subject invention utilize a sodiumphosphotungstate reagent for reacting with a urine sample. There areseveral types phosphotungstate that can be utilized with the subjectinvention. For example, lithium phosphotungstate could also be used inthe embodiments of the subject invention. Preferably, the selectedphosphotungstate that is utilized is molybdate-free. A person with skillin the art, having benefit of the subject disclosure, would be able todetermine any one of several types of phosphotungstate that could beused in the reagent of the subject invention.

In one embodiment of the method of the subject invention, a urine sampleis analyzed to obtain a first Uric Acid Equivalents value for the sampleutilizing the reagent of the subject invention. However, if the testresults show a Uric Acid Equivalents value that is not initiallyindicative of the presence of an oxidative-adulterant, analysis with theother assays of subject invention may warrant confirmatory testing. Inthat situation, the sample can be sent to a confirmatory laboratory forfurther testing. One embodiment of the method of the subject inventionincludes the confirmatory laboratory conducting further tests of themarkers to obtain a second or additional Uric Acid Equivalents values.If the test results of a confirmatory laboratory do not indicate orcorroborate adulteration, the one or more Uric Acid Equivalents valuescan then be compared to the initial Uric Acid Equivalents value obtainedby the screening laboratory. All of the Uric Acid Equivalents values,first, second, and any subsequent values, can be plotted to create anOHist. If the OHist confirms that the levels of Uric Acid Equivalentsvalues decreased during the time window of the sample, which is from thetime the sample was obtained to the time of the confirmatory testing,then the presence or historical presence of an oxidative-adulterant canbe definitively confirmed.

One embodiment of a phosphotungstate reagent can be prepared as follows:

Reagent 1: Phosphotungstate Reagent—1 Liter: Add 27 grams of molybdenumfree sodium tungstate to approximately 333 mL deionized water in areflux vessel. Add 30.9 grams of phosphoric acid. Add boiling chips andreflux gently for 2 hours. Cool to room temperature and then dilute to 1Liter with deionized water. Add 21.0 grams of Lithium Sulfate and mix.

All ingredients are ACS grade. Molybdenum free Sodium Tungstate wasobtained from GFS Chemicals, Powell Ohio 43065. The reagent so preparedis stable for at least 1 year refrigerated at 2-8 degrees Celsius.

When utilized in an automated analyzer of the type used for urineanalysis, the following additional buffer reagent and calibrator reagentcan be formulated for use in such equipment:

Reagent 2: Carbonate Buffer Reagent—1 Liter. Dissolve 119 grams of ACSgrade sodium carbonate to 800 mL of deionized water. Add 7.5 gramssodium hydroxide. Add deionized water to bring the volume to 1 Liter.This reagent is used to buffer the test sample and blank.

The reagent is stable for at least 1 year at ambient temperature 20-30degrees Celsius.

Reagent 1 and 2 are stable for at least 1 year when packaged togetherand stored at 2-8 degrees Celsius.

Reagent 3: Uric acid 40 mg/dL Calibrator: 1 Liter—Dissolve 0.63 grams ofACS grade lithium carbonate in 500 mL of Deionized water. Warm to about45 degrees Celsius. The mixture should not be warmer than 50 degreesCelsius for the next step. Add 400 mg of ACS grade uric acid and mixuntil dissolved. Dissolve 0.5 grams of sodium azide and then dilute to 1liter with deionized water. This calibrator is stable for at least 1year refrigerated at 2-8 degrees Celsius.

One embodiment of the method utilizing the reagents 1, 2, and 3, inautomated equipment is as follows:

-   -   Manual preparation of final reagent:        -   Test—Add 100 μL of Urine Sample to 2 mL of Phosphotungstate            Test Reagent 1 and mix.        -   Reagent Blank—Add 100 μL of Deionized water to 2 mL of            Phosphotungstate Reagent 1 and mix.        -   Add 1 mL of Carbonate Buffer Reagent 2 to Reagent Blank and            Test Reagent and mix each one separately.        -   Incubate for 5 minutes at 37 degrees Celsius.        -   Measure absorbance at 700 nm of Reagent Blank and Test            Reagent.        -   Calibrator absorbance is developed by using the calibrator            as a Test.        -   The Test Net absorbance (Test Absorbance—Blank Absorbance)            is used to calculate the Uric Acid Equivalent result as            follows:            (Test Net Absorbance/Calibrator Net Absorbance)×Calibrator            Absorbance=Value of Calibrator

The absorbance measurement may be made at 600-700 nm. No secondarybichromatic wavelength should be used.

Following are specifications for testing a urine sample with a MindrayBS-200 automated chemical analyzer. These settings are intended asguidelines and those with skill in the art would recognize that theparameters can vary between instruments.

Test: OX Hist No. User Defined Full Name: Oxidant History Reaction Type:Endpoint Pri. Wave 670 nm Sec Wave none Direction: Increase Reac. Time:0 and 11 Incubation Time:   3 Unit: Mg/dL UAEq Precision: Integer R1:180 R2:  90 Sample Volume;  10 Mixed Reagent Blank: Optional Compensate:Slope :1 Intercept: 0

Following are specifications for testing a urine sample with theBeckman-Coulter AU 400, AU 400e, AU 480, AU 640, AU640e and AU680 Seriesautomated chemical analyzers. These settings are intended as guidelinesand those with skill in the art would recognize that the parameters canvary between instruments.

Reagent ID: User defined Test Name: Oxidant History Sample Volume:  10R1 Volume: 100 R2 Volume:  50 Wavelength: Pri: 700 Sec. None Method: +Reaction Slope: POS Measuring Point 1: First  25 Last 27 Measuring Point1: (Not Applicable) Calibration Type: AA Formula: Y = AX + B

The reagents and methods described above can be used to exploit thekinetics of Uric Acid Equivalents measurement by performing a screeningassay on a urine sample soon after collection and then repeating thescreening assay hours or even days following the first test. Forexample, the collecting site or first immunoassay screening laboratorycan test the sample and obtain an initial Uric Acid Equivalents valuefor the sample. If the initial value concludes the possible presence ofan adulterant, the results can be reported to the confirmatorylaboratory. The confirmatory laboratory can also obtain one or more UricAcid Equivalent values on the same sample at a later time. The resultsof the initial Uric Acid Equivalents value can be compared with theresults of the one or more Uric Acid Equivalents values obtained laterby the confirmatory laboratory and plotted to obtain an OHist. Ifanalysis of the OHist shows a reduction in the Uric Acid Equivalentsvalues over time, the presence of an oxidative-adulterant can beconsidered definitive proof of the use of an oxidative-adulterant in thesample. Even if the analysis of the Uric Acid Equivalents values do notfall below the mandated cutoff level indicative for positiveadulteration, adulteration can still be definitively proven if the UricAcid Equivalents values obtained from confirmatory laboratory tests havefallen significantly during the time window of the sample.

II. Two Assays for Detecting Counterfeit Urine Utilizing Short-Duration(SD) Acid Phosphatase (AP) and Long-Duration (LD) Alkaline-Phosphatase(ALP) Markers in Urine

Embodiments of a Counterfeit Urine assay of the subject invention areunique in their ability to detect the absence of a constituent in theurine sample, rather than the presence of a constituent, as anindication that the sample is not true human urine. U.S. patentapplication Ser. No. 15/961,003, filed on Apr. 24, 2018, disclosesassays and methods of use that can be utilized with embodiments of thesubject invention to detect counterfeit urine. The entirety of U.S. Ser.No. 15/961,003 is hereby incorporated by reference.

Embodiments of the SD and LD counterfeit urine assays of the subjectinvention employ two different markers normally found in urine.Advantageously, the markers utilized with the testing methodology of thesubject invention are activated under significantly different conditionsand detectable under similar conditions. A further advantage of thesemarkers is their labile nature that makes them impractical to use asadditives in counterfeit urine products. Furthermore, while efforts canbe made to mimic the presence of these markers, such as by addition ofindicator dyes, the embodiments of the subject invention can detect suchefforts.

Specifically, embodiments of the subject invention utilize the labilemarkers acid phosphatase (AP) and alkaline phosphatase (ALP) to detectwhether urine is “true urine,” being of human origin, or is acounterfeit urine product. AP and ALP are preferred enzyme markersbecause they are present in urine produced by both males and females andhave poor in vitro stability. These markers are temperature sensitive,which makes them unstable after sample collection. For example, AP is ashort-duration (SD) marker, often degrading in a sample within 3 days.In contrast, ALP is a long-duration (LD) marker, often degrading inabout a week to 10 days. Samples are usually tested within a few daysafter collection. If AP in the sample has degraded before testing, it isstill likely at least some ALP will remain in the sample and can provideresults.

In a further embodiment, the subject invention utilizes the chromogenicsubstrates thymolphthaleine monophosphate and p-nitrophenyl phosphate,which are catalyzed by AP (at pH 4-6) and ALP (at pH 8-10),respectively. Thymolphthalein monophosphate and p-nitrophenyl phosphateare advantageous because they are catalyzed at significantly differentpH levels, but produce chromogens that are activated under identicalalkaline conditions. The subject invention utilizes these advantageouscharacteristics to create a single test control for detecting thepresence of both of these chromogens.

Advantageously, these markers have a labile nature that makes themunsuitable, or at least impractical, as additives to counterfeit urineproducts. The constituents are temperature sensitive causing them todegrade within a few days and become undetectable, often before thecounterfeit urine product can be used. The reagents according to thesubject invention are safe and non-toxic.

The substrate thymolphthalein monophosphate is shown below:

Thymolphthalein monophosphate is catalyzed by AP at betweenapproximately pH 4 to approximately pH 6. When combined with an aliquotof a sample of urine, AP in the urine hydrolyzes this substrate therebyproducing free thymolphthalein. Thymolphthalein is a colorless productat the acid pH necessary for hydrolysis. When exposed or subjected toalkaline conditions, thymolphthalein exhibits a blue color.Thymolphthalein monophosphate is catalyzed to form thymolphthalein asfollows (Equation I):

Another substrate utilized with embodiments of the subject invention isp-nitrophenylphosphate (shown in the acid form):

P-nitrophenyl phosphate is catalyzed by urinary alkaline phosphatase(ALP) at between approximately pH 8 and approximately pH 11.P-nitrophenyl phosphate is also catalyzed by urinary acid phosphatase atbetween approximately pH 5 and approximately pH 6. When added to asample of urine, alkaline phosphatase (ALP) in the urine hydrolyzes thep-nitrophenyl phosphate substrate, thereby producing free p-nitrophenol.The p-nitrophenol turns yellow at the pH necessary for hydrolysis, thusis self-indicating. Acid phosphatase (AP) also catalyzes the hydrolysisof p-nitrophenol phosphate to liberate p-nitrophenol, which under theacid is colorless at the acid pH required for this AP reaction. Thus, asecond step that alkalinizes the solution is necessary to activatep-nitrophenol as a chromagen that indicates the hydrolysis ofp-nitrophenyl phosphate.

v-nitrophenylphosphate is catalyzed by ALP as follows (Equation II):

Thus, both thymolphthalein and p-nitrophenol are chromogens that arecolor-activated under alkaline conditions, such that thymolphthaleinturns blue and p-nitrophenyl turns yellow.

Automated analyzers typically utilize liquid reagents. In oneembodiment, the reagents of the subject invention are formulated asliquids for use in an automated analyzer. When a sample treatedaccording to the subject invention is analyzed spectrophotometrically,the blue color produced by thymolphthalein is absorbed at a wavelengthof approximately 600 nm. The yellow color produced by p-nitrophenol isabsorbed at a wavelength of between approximately 405 nm andapproximately 410 nm.

The stability of the thymolphthalein monophosphate and p-nitrophenylphosphate substrates makes them useful for dip stick or spot tests. Spottests are well-known in the art and can have a liquid, semi-solid, orsolid transport medium. For the sake of providing a visualrepresentation of the embodiments of the subject invention, referencewill be made to a dipstick methodology, as shown in FIGS. 5, 6, and 7.The ability to formulate the substrates of the subject invention fordipstick spot tests is known in the art and will not be described indetail here.

Embodiments of the subject invention utilize reagent systems, whichinclude substrate reagents that are catalyzed by either ALP and/or APand a color-developer reagent that activates or enhances the appearanceand color of the resulting chromogen. The reagent system embodiments canalso utilize a control reagent, which advantageously confirms thepresence or absence of one or both of the chromogens.

One embodiment of a reagent system includes a substrate reagentcomprising the substrate p-nitrophenyl phosphate that is buffered tobetween approximately pH 9.5 to approximately pH 11, which promotesformation of the chromogen in the presence of the catalyst. In a furtherembodiment, the reagent system includes a control reagent, whichcomprises no p-nitrophenol or p-nitrophenyl phosphate. The controlreagent can also comprise all or most of the constituents of thesubstrate reagent, except for the substrate and the chromogen. In a yetfurther embodiment, the p-nitrophenyl phosphate reagent system has analkaline color-developer reagent buffered to between approximately pH9.5 to approximately pH 11. The color-developer reagent can be utilizedwith the control to determine whether a sample is true urine or acounterfeit urine product.

It can be seen in FIG. 5 an example wherein the p-nitrophenol phosphatereagent system is illustrated with a dip stick method. In the dip stickexample shown in FIG. 5, the upper Test spot comprises the substratereagent and the lower Control spot comprises the control reagent, whichare, initially, colorless. When the spots are saturated with a sample,several reactions can occur, depending upon the constitution of thesample. True urine causes the upper Test spot to turn a distinctiveyellow color, by the reaction of the ALP in the urine with thep-nitrophenyl phosphate, which cleaves the phosphate moiety, leaving thechromogen p-nitrophenol. When saturated with true urine, the Controlspot remains colorless or can appear to turn the same color of thesample. The Control spot, at this point, does not have the same yellowcolor as the Test spot. The addition of other ingredients to a samplecan cause the same result as true urine, i.e., turn the Test spotyellow. Advantageously, the color-developer reagent of the subjectinvention can detect these other potential additives, if present.

With regard to FIG. 5, to confirm whether the constitution of the sampleis true urine or a counterfeit urine product, the Control spot istreated with the color-developer reagent of the p-nitrophenyl phosphatereagent system, which changes the Control spot to an alkaline pH. If,after addition of the color-developer reagent, the Control spot turnsthe same or a similar yellow color as the Test spot, it indicates thatthe p-nitrophenol chromogen was likely added to the sample, whichfurther indicates the sample being a counterfeit urine product or was atleast subject to other tampering and, thus, invalid.

A further advantage of this embodiment of the p-nitrophenyl phosphatereagent system, is that it is also possible to detect the presence ofthymolphthalein that may have been added to the sample. If the Test spotand/or the Control spot turn blue, as shown in FIG. 5, it indicates thatthymolphthalein was added to the sample, which reacts, at least mildly,in the alkaline pH of the Test spot, such that both spots are imbuedwith a blue color, thus, indicating invalidity of the sample. Additionof the second, alkaline color-developer reagent to the Control spot canturn it blue, providing further indication that thymolphthalein waslikely added to the sample, as indicated in FIG. 5, thus, anotherindication of invalidity of the sample. If the Test spot and/or theControl spot turns green, both p-nitrophenol and thymolphthalein wereadded to the sample, whereby the color transformations of the yellow andblue chromogens combine to forms the green color, as also shown in FIG.5, again, indicating that the sample is invalid.

One embodiment of a Reagent System for detection of p-nitrophenol usingautomated equipment is described below:

Substrate Reagent: Alkaline Buffered and a combination of the followingComponent A and Component B.

Component A: 31 grams 2-amino-2-methyl-1-propanol (AMP) 0.2 gramsProclin 300 0.6 grams N-hydroxyethylene-diaminetriacetic acid (HEDTA)Combine the above ingredients with approximately 800 ml deionized waterand mix to dissolve to form solution #1. 0.3 grams Zinc Sulfate•7H₂O Addto solution #1 and mix to dissolve to form solution #2. 0.4 gramsMagnesium Acetate•4H₂O Add to solution #2 and mix to dissolve to formsolution #3. Adjust pH of solution #3 to 10.2 +/− 0.05 using 6N HCl, toform solution #4. 4 grams Brij 35 30% solution Add to solution #4 andmix to dissolve to form solution #5. Reagent #1 is formed by addingdeionized water to solution #5 to make 1 Liter and mix to dissolve.Avoid excess exposure to air following pH adjustment. Note: This is abuffered solution. Component B: 900 mL Deionized water 0.5 grams Proclin300 2.0 grams Imidazol 20 grams p-nitrophenol phosphate disodium salt(hydrate form) CAS No. 123359-43-3 Adjust to pH 6.5 +/− 0.01 with pure 6N HCl and bring volume to 1000 ml with deionized water. Control Reagent:Component A is replaced with deionized water and an alkaline solution,such as Component B of Example 1 above, is added as in the test. Thistype control is only required for dip-stick tests or where the reagentis measured as an end- point assay. If the p-nitrophenol phosphatereagent is detected by an automated device as a fixed time or kineticassay, the initial absorbance of the reagent may be observed as acontrol and limits set for the initial absorbance if permitted by theautomated device. Otherwise, a control as provided above must beperformed to detect addition of adulterants to the counterfeit urine.Calibrator: 100 Units/L Urinary tract Protein-LD 1.0 Liter 50% Glycerolin Deionized water 1.6 grams Tris Hydrochloride 0.3 grams MagnesiumChloride 0.02 grams Zinc Chloride To the above solution add 50 Units ofAlkaline phosphatase CAS RN 9001-78-9. Stock Calibrator: 140 mgp-nitrophenol 0.2 g Proclin 300 300 mg Imidazol

Combine with approximately 800 mL of deionized water and mix todissolve. Adjust pH to 6.5 using diluted HCl. Final Calibrator is StockCalibrator diluted 1:250 with deionized water to obtain a FinalCalibrator solution having the equivalent of 100 Enzyme Units (UrinaryTract Protein). Note: An enzyme unit (U) is specific to particularenzyme and is defined as the amount of the enzyme that catalyzes theconversion of 1 micro mole of substrate per minute. Following arespecifications for testing a urine sample with a Mindray BS-200automated chemical analyzer. These settings are intended as guidelinesand those with skill in the art would recognize that the parameters canvary between instruments.

Test: UALP No. User Defined Full Name: Alkaline Phosphatase ReactionType: Fixed-time Pri. Wave 510 nm Sec Wave 630 Direction: Increase Reac.Time: 0 and 2 Incubation Time: 20 Unit: UALP Units Precision: IntegerR1: 180 Sample Volume; 46 Mixed Reagent Blank: Compensate: Slope: 1Intercept: 0 Note: The control procedure for the above is the same asthe test procedure, but uses the control reagents. The calibration forthe above is the same as the test procedure.

Following are specifications for testing a urine sample with the BeckmanCoulter AU 400, AU 400e, AU 480, AU 640, AU640e and AU680 Seriesautomated chemical analyzers. These settings are intended as guidelinesand those with skill in the art would recognize that the parameters canvary between instruments.

Reagent ID: User defined Test Name: True Urine LD Sample Volume:  35 R1Volume: 120 R2 Volume:  30 Wavelength: Pri: 405 Sec. — Method: FIXEDTIME Reaction Slope: POS Measuring Point 1: First 13 Last 27 Units: UTPUnits Calibration Type: AA Formula: Y = AX + B Point 1 CONC: 0 Point 2CONC: 100 Note: The control procedure for the above is the same as theTest procedure, but uses Control Reagent #1.

In another embodiment, a substrate reagent comprises thymolphthaleinmonophosphate and is buffered to between approximately pH 4 andapproximately pH 6. This reagent reacts with AP to promote the formationof the chromogen thymolphthalein. At this pH, there will typically be noformation of the color of the chromogen. In a further embodiment, thethymolphthalein monophosphate reagent system has a color-developerreagent having an alkaline pH of between approximately 9.5 toapproximately 11. This can be similar to the color developer reagentutilized with the p-nitrophenyl phosphate substrate reagent, describedabove. In one embodiment, the control reagent utilized with thisembodiment of the reagent system can comprise all or most of theconstituents of the substrate reagent, but no thymolphthaleinmonophosphate substrate, nor any thymolphthalein, which is the chromogenformed from p-nitrophenyl phosphate when catalyzed with AP.

FIG. 6 illustrates a non-limiting example of a dip stick on which thesubstrate reagent and the control reagent have been stabilized ontospecific test spots on the dipstick. In one embodiment, the firstsubstrate reagent is stabilized in a fashion that will provide thenecessary acidic pH for the substrate to be catalyzed upon addition oftrue urine. It will be understood by a person skilled in the art thatthis reagent system can be in liquid form and the method can be utilizedwith automated chemical analyzers. With regard to FIG. 6, which shows anon-limiting example of a dip stick method, the upper Test spotcomprises the substrate reagent and the lower Control spot comprises thecontrol reagent, which are both initially colorless. When the spots aresaturated with a sample, several reactions can occur depending upon thenature of the sample. True urine causes the Test spot to turn a bluecolor, due to reaction of the AP in the urine with the p-nitrophenylphosphate, which cleaves the phosphate moiety, leaving the chromogenp-nitrophenol. When saturated with true urine, the Control spot remainscolorless or appears as the same color as the sample, e.g., pale yellow,but does not have the same blue color as the Test spot. However, theaddition of other ingredients to a sample can cause the same results astrue urine. Advantageously, the color-developer reagent of the subjectinvention can detect whether these other ingredients have been added tothe sample.

With regard to FIG. 6, when the Test spot and Control spot are treatedwith the color-developer reagent of the reagent system, the Test spotand Control spot are changed to an alkaline pH, thereby activating thecolor change of the thymolphthalein to blue. If, after addition of thecolor-developer reagent, the Test spot turns blue and the Control spotremains colorless, it indicates that the sample was true urine, valid,and thymolphthalein liberated from the thymolphthalein monophosphatesubstrate was present on the Test spot. If, after addition of thecolor-developer reagent, neither the Test spot, nor and the Control spotturn blue, it indicates that the sample did not contain AP to react withthe thymolphthalein monophosphate substrate and was invalid. This canindicate that the sample was a counterfeit urine product or wasotherwise subject to tampering. Counterfeit urine products or othertampering of the sample can also be suspected if, after addition of thecolor-developer reagent, the Control spot turns the same or a similarblue color as the Test spot, an indication that the thymolphthaleinchromogen was added to the sample.

Furthermore, with this embodiment of the reagent system, it is possibleto detect the presence of p-nitrophenol that may have been added to thesample. If, after addition of the color-developer reagent the Test spotand/or the Control spot turn yellow, as shown in FIG. 6, it can be anindication that p-nitrophenol was likely added to the sample. If theTest spot and/or the Control spot turns green, it can be an indicationthat both p-nitrophenol and thymolphthalein were likely added to thesample, whereby the color transformations of the yellow and bluechromogens combine to form the green color, as also shown in FIG. 6.FIG. 7 illustrates a comparison of the AP and ALP assays bydemonstrating the color effects on a dipstick embodiment that includesboth assays.

One embodiment of a Reagent System for detection of thymolphthaleinusing automated equipment is described below:

Substrate Reagent: comprises Component A and Component B, which areadded in a 50/50 ratio to obtain the final concentration:

Component A:. 17.68 grams Sodium Acetate, added to approximately 400 mLdeionized water and mix until dissolved 1.6 grams Citric Acid followedwith sufficient Acetic Acid (redistilled) to adjust pH to 6.0 @ 25° C.0.2 grams Proclin 300 Add deionized water to obtain final volume of 500mL Component B: 5.0 grams Brij 35 detergent, added to 500 mL deionizedwater and mix until dissolved 1.0 grams Thymolphthalein monophosphatesodium salt and mix until dissolved Combine equal parts of Component Aand Component B to obtain a final solution of Substrate Reagent. Forexample, 500 mL of Component A can be combined with 500 mL of ComponentB to obtain 1 liter of Substrate Reagent. The Substrate Reagent shouldbe stored refrigerated at between 4°-8° C., away from light.Color-Developer reagent: 20 grams Sodium Hydroxide 53 grams SodiumCarbonate (Anhydrous) Combine with 1 liter of deionized water to obtainfinal concentration and volume. Control Reagent: 500 mL of deionizedwater and 500 mL of Component B added. Calibrator: 100 Units UrinaryTract Protein 700 mL n-propanol 300 mL Deionized water 20 mgThymolphthalein

The Urinary Tract Protein Units are arbitrary and based upon the amountof thymolphthalein produced during the time of incubation of Component Awith a given ratio of component A to urine. Thus, if the time ofincubation is doubled, the value of the calibrator can be cut in half.If the urine volume to Component A is lowered by decreasing samplevolume, the value can be increased proportionately. The arbitrary unitsare intended to avoid traditional enzyme measurement and the marker(s)are to be described as Urinary Tract Glyco-proteins, so as to obscurethe identity of subversion additions to the counterfeit urine.

Following are specifications for testing a urine sample with a MindrayBS-200 automated chemical analyzer. These settings are intended asguidelines and those with skill in the art would recognize that theparameters can vary between instruments.

Test: True Urine SD No. User Defined Full Name: True Urine ReactionType: Fixed-time Pri. Wave 578 nm Sec Wave 670 Direction: Increase Reac.Time: 0 and 2 Incubation Time:  20 Unit: UTP Units Precision: IntegerR1: 180 R2:  40 Sample Volume:  46 Mixed Reagent Blank: Compensate:Slope: 1 Intercept: 0

Following are specifications for testing a urine sample with the BeckmanCoulter AU 400, AU 400e, AU 480, AU 640, AU640e and AU680 Seriesautomated chemical analyzers. These settings are intended as guidelinesand those with skill in the art would recognize that the parameters canvary between instruments.

Reagent ID: User defined Test Name: True Urine Sample Volume:  30 R1Volume: 123 R2 Volume:  43 Wavelength: Pri: 600 Sec. 700 Method: FIXEDTIME Reaction Slope: POS Measuring Point 1: First  14 Last  16 MeasuringPoint 1: (Not Applicable) Calibration Type: 2AB Formula: Polygonal Point1 H20 CONC: 0 Point 2 Cal CONC: 100 Note: the control procedure is thesame as the Test procedure.III. Specific Gravity Assay Utilizing Sodium (Na+) and Potassium (K+)Markers in Urine

The subject invention provides methods and reagents useful for analysisand measurement of specific constituents in a urine sample that can beused to derive a Specific Gravity Index (SGI) for a urine sample. Whenthe SGI of a given urine sample is compared to the SGI of known normalurine samples, results can be used to determine whether the given samplewas adulterated. U.S. patent application Ser. No. 15/651,334, filed Jul.17, 2017, discloses assays and methods of use that can be utilized withembodiments of the subject invention to detect adulteration of a urinesample. The entirety of U.S. Ser. No. 15/651,334 is hereby incorporatedby reference.

Specific gravity of a liquid is a measure of the weight of a liquiddivided by the weight of water of equal volume. The constituents foundin the highest concentrations in urine are sodium chloride and potassiumchloride and, as such, contribute most to the specific gravitymeasurement for a urine sample. However, there are other constituents,such as urea, which also contribute to the overall specific gravity of aurine sample. A Specific Gravity Index, (SGI), according to the subjectinvention, is a measurement obtained by utilizing a subset of theconstituents found in a urine sample. More specifically, the SGI is ameasurement of the weight of the non-aqueous subset of constituents ofthe sample per unit volume. Advantageously, the subset of constituentscan also be used as markers, according to the subject invention, forautomating analysis of a urine sample, so as to obtain a SG1.

One embodiment of the subject invention utilizes the sodium (Na+) andpotassium (K+), naturally found in a urine sample, as markers. In afurther embodiment, these same markers are used to obtain a SGI for thesample. In a specific embodiment, a sodium-potassium dependentβ-Galactosidase is utilized along with an indicator chromogen ofo-nitrophenylgalactoside (o-NPG). In one embodiment, the method of thesubject invention results in the formation of a yellow color due tocleavage of the o-NPG into o-nitrophenol, a molecule that can beanalyzed by the spectrophotometry methods utilized in most clinicalanalyzers to obtain a SGI for the given sample. The chromogen can becleaved to o-nitrophenol by using the sodium or potassium activatedβ-Galactosidase. Thus, the amount of sodium and/or potassium in a givenurine sample can dictate the amount of cleaved o-NPG created by thereaction. A measurement of both sodium and potassium, can beextrapolated to yield a total mEq/L of both substances in the sample Ina specific embodiment, the reagent and method of the present inventionemploy sodium-potassium dependent beta-galactosidase (β-galactosidase)in conjunction with an indicator chromogen of o-nitrophenylgalactoside(o-NPG). The reaction causes the chromogen to be cleaved intoo-nitrophenol by the sodium and/or potassium activated β-galactosidase.Advantageously, the rate at which the yellow o-nitrophenol is producedfrom the colorless o-NPG can be measured spectrophotometrically at aprimary wavelength of 405-410 nm. FIG. 8 illustrates this reaction. Therate of increase in absorbance at 405 nm-410 nm is proportional to totalsodium and potassium concentration in the sample. Colorimetricmeasurements outside a known normal range for a SGI of urine can be anindication of abnormal levels of sodium and/or potassium in the sample.This can be an indication that the sample integrity has been compromisedand the sample is not valid.

FIG. 10 illustrates the sensitivity of the SGI assay of the subjectinvention. The typical tests used for detecting a non-normal specificgravity, such as “dipsticks” that rely upon the amount of sodium, areusually not effective where the w/v of adulterant in a sample is below10%. FIG. 9 demonstrates that embodiments of the subject invention arecapable of detecting adulteration of only 5% w/v and even as little as1% w/v, for the most common adulterants.

Sulfhydryl blocking adulterants such as iodoacetamide, at aconcentration of 2 mM/L, have been shown to be effective in subversionof at least one of the most widely used EIA tests, but are detectablewith the Specific Gravity Index assay of the subject invention. Cationicdetergents are also being used to subvert standard assays. Cationicdetergents are found in common over-the-counter eye drops, which have aconcentration of about 0.05%, which is effective for subversion. TheSpecific Gravity Index assay is useful for detecting this type ofsubversion. As will be discussed below, both of these two new types ofsubversion can cause a Specific Gravity Index to fall below 1.003 andnear that of water. Furthermore, the use of heavy metal adulteration,such as with cadmium acetate, copper sulfate, lead acetate, silvernitrate, mercuric chloride, chromium VI, and p-chloromercuribenzoate, at2.0 mMol/L, can also lower the Specific Gravity Index to below 1.003 andnear that of water.

As mentioned above, in one embodiment the reagent used in this SGI assayof the subject invention produces a yellow coloration to the urinesample being tested. More specifically, the cleavage of theo-nitrophenylgalactoside (o-NPG) into o-nitrophenol can produce a yellowcoloration. The amount of coloration imparted to the sample is dependentupon the amount of sodium and potassium present in the sample toactivate the β-galactosidase. Therefore, it is possible to utilize highurine:reagent ratio to ensure that the SGI for a sample is notinadvertently truncated due to insufficient reagent. In one embodiment,the sample:reagent ratio is between approximately 1:20 to approximately1:100. In a more specific embodiment, the sample:reagent ratio isbetween approximately 1:50 and approximately 1:80.

Utilizing these percentages, the range for a normal SGI can becalculated as being between approximately 32 mEq/L and approximately 364mEq/L. Thus, a combined Total Sodium and Potassium Value, which is thetotal amount of sodium and potassium in a urine sample, yielding a SGIbelow 32 mEq/L can be indicative of sample adulteration by dilution anda SGI over 364 mEq/L can be indicative of some other adulteration orsubstitution technique.

Specific gravity is a dimensionless quantity. As such, the mEq/L unitsof total sodium and potassium when converted gives a SGI of 0.003 forthe 32 mEq/L low-end cut-off value reagent calibrator and a SGI of 0.035for the 264 mEq/L high end cut-off value reagent calibrator. Whenmeasuring specific gravity, the decimal value is the specific gravity ofa substance minus the specific gravity of H₂O, which should be 1.0000.Embodiments of the subject invention provide a Specific gravityEquivalent Value, based on the substituents of sodium and potassium in aurine sample, which can be calculated by adding 1 to the decimal valueof the SGI. The currently recommended low specific gravity limit is1.003 and the recommended high specific gravity limit is 1.035. Thus,for the purposes of automation and recording, it can be necessary toconvert the SGI by adding 1.0 to the decimal value.

Alternatively, SOI can be calculated as the weight per unit volume ofthe non-aqueous constituents of a sample divided by the specific gravityof water, which is 1.0000 mg/mL. Thus, when the combined Total Sodiumand Potassium Value is used, the SGI can range from betweenapproximately 0.0030 and approximately 0.0350. A SGI below 0.0030 can beindicative of dilution of the sample and a SGI above 0.0350 can beindicative of adulteration.

As mentioned above, embodiments of the subject invention can beincorporated with automated laboratory equipment that is typically usedfor urine sample aliquot analysis. In one embodiment, theβ-galactosidase and the o-NPG can be formulated as a single reagent,such that the reaction can be conducted as a single step. In analternative embodiment, the 3-galactosidase and the o-NPG can beformulated as separate reagents, such that the reaction is carried outin two or more steps.

Potassium chloride and sodium chloride, in the molar concentrationsdiscussed above, can be formulated in an aqueous solution by a person ofskill in the art to obtain an appropriate calibrator. In addition, 0.2%ProClin™ 300 can be used as a stabilizer for the aqueous calibrator. Thestabilized calibrator can be used with automated machinery, such asclinical analyzers, to calibrate the high end cut-off value of 264 mEq/Land can be appropriately diluted to also calibrate the low end cut-offvalue of 32 mEq/L. Thus, a urine sample with an SGI above or below thisrange can be considered adulterated or tampered with in some manner.

One embodiment of the subject invention provides a liquid regent thatcan be added to a urine sample to initiate the yellow color change inthe sample aliquot. The formulation for the reagent is prepared asfollows:

Reagent Concentrations: beta-Galactosidase 25 to 8000 U/L ortho-NPG >0.2mM buffer pH 7-9.5 Mg²⁺ 0.01-10 mmol/L EGTA (free acid) 1-20 mmol/LSerum Albumin 0-5 g/L N-Acetyl Cystine 0.05-2M ProClin 300 ® 2 grams/L

The ingredients should preferably be salt free, particularly with regardto heavy metals, calcium, sodium and potassium. It can also be desirablefor pH adjustments to be made on aliquots of the reagent. Ideally, suchaliquots are discarded in order to minimize potassium contamination ofthe reagent.

Urine typically contains calcium, which can vary between samples.Calcium can be a competitive inhibitor of the activation ofbeta-galactosidase by magnesium also present in the urine. Calcium isalso unstable and can affect stability of the reagent. In oneembodiment, EGTA is utilized in a reagent of the subject invention tocomplex calcium in the urine sample. The amount necessary will dependupon the amount of calcium that needs to be deactivated in a givensample. In the embodiment shown above, approximately 0.5-20 mmol/L areutilized. It is within the skill of person trained in the art todetermine the appropriate amount of EGTA that may be required for aparticular sample. Such variations that provide the same functionality,in substantially the same way, with substantially the same result arewithin the scope of this invention.

In an alternative embodiment, which can be useful in automationequipment, the ortho-nitrophenylgalactoside (o-NPG) can be provided as asecond reagent of known concentration that can be added to the firstregent to achieve the desired final concentration indicated above.

The stability of the o-NPG containing second reagent can be maximized byadjusting pH to be about 6.5. Ideally, a minimal amount of buffer isused to achieve this pH, so that when the second reagent is added to thefirst reagent, there is minimal or no effect on the final reaction pH,which should be about 8.5.

The addition of magnesium to the calibrators and controls in proportionto their concentration relative to mean normal Specific Gravity Indexcan improve sensitivity to the effect of dilution, or measurements atthe lower cut-off value of 0.0030 SGI. In one embodiment, the amount ofmagnesium utilized in a reagent of the subject invention is betweenapproximately 0.01 mmol/L to approximately 0.01-2 mmol/L. In a moreparticular embodiment, the amount of magnesium utilized in a reagent ofthe subject invention is between approximately 0.01 mmol/L toapproximately 1 mmol/L.

Prior to analysis, the analyzer can be calibrated. This can be done witha reagent blank, a low end calibrator having a sodium and potassiumconcentration that is at or near the normal low range limit in humanurine and the high end calibrator having a sodium and potassiumconcentration that is at or near the normal high range limit in humanurine. In one embodiment, the low end calibrator contains approximately:20 mEq/L of sodium chloride and 11.7 mEq/L of potassium chloride. In afurther embodiment, the high end calibrator contains approximately 230mEq/L of sodium chloride and 134 mEq/L of potassium chloride.

The measurement of creatinine levels in a urine sample is currentlyaccepted as the gold standard for determining whether a sample has beendiluted. Current government regulations mandate that the cut-off levelfor determining whether a sample has been diluted is 20 mg/dL. Thus, anysample presented that is measured with a creatinine level below 20 mg/dLis considered compromised by dilution.

Samples having a below-normal creatinine level can be further tested bymeasuring the specific gravity of the sample. Current tests for specificgravity are determined by measuring the uric acid levels in a sample andextrapolating a value from that measurement. If the specific gravity ofa sample is measured to be below 1.0030, the sample is deemed as beingabnormal or having been subjected to tampering.

Following are specifications for analyzing urine samples with severaldifferent types of automated equipment, including the Mindray BS-200 andthe Beckman Coulter AU 400, AU 400e, AU 480, AU 640, AU640e and AU680Series Clinical Chemistry Analyzers. The settings shown are intended tobe guidelines for the indicated instruments. It is within the skill of aperson trained in the art to recognize that such parameters will varybetween instruments.

Assay Parameter Settings for Mindray BS-200 Analyzer

Test: SGI No. User Defined Full Name: Specific Gravity Index ReactionType: Fixed-time Pri. Wave 405 nm Sec Wave 510 nm Direction: IncreaseReac. Time: 0 and 9 Incubation Time:   3 Unit: g/mL Precision:   0.0001R1: 250 R2: 100 Sample Volume;   4 Compensate: Slope: 1 Intercept: 1.0Calibration Parameters Rule Logit-Log 5P Replicates   1 Determinationcoeff.   0Calibrators: 0.0000 (deionized water), 0.0030 (low cut off fordilution), a mid-calibrator of 0.019, a high cut-off calibrator forsalting of 0.0350, and a high range calibrator of 0.0500Assay Parameter Settings for Beckman Coulter AU 400, AU 400e, AU 480, AU640, AU640e and AU680 Series Clinical Chemistry Analyzers

Reagent ID: User defined Test Name: Specific Gravity Index SampleVolume: 2 R1 Volume: 107 R2 Volume: 43 Correlation factor A 1.0 B 1.0Wavelength: Pri: 410 Sec. 600 Method: FIXED Reaction Slope: + MeasuringPoint 1: First 11 Last 26 Measuring Point 2: (Not Applicable)Calibration Type: 5AB Formula: Polygonal Counts 1 CONC Point 1 H200.0000 Point 2 Low C/O 0.0030 Point 3 MID 0.0190 Point 4 Hi C/O 0.0350Point 5 Hi Range 0.0500IV. Reagent System for Measurement of Creatinine in a Urine Sample

Most laboratories that conduct DOA urine tests currently performcreatinine assays as a means of detecting invalid samples. If thecreatinine of a sample is measured below 20 mg/dL, procedures indicatethat specific gravity tests should then be performed on the sample.Although measurement of creatinine has historically been the goldstandard for the detection of subversion by dilution, new subversiontechniques, such as in vivo dilution, are now being used to subvertdetection. The technique of in vivo dilution was first discovered instudies of athletes and body builders who often take, a.k.a. “load”,supplemental creatine to increase muscle mass. Creatine is converted inmuscle to creatinine, which is subsequently excreted in the urine.[Schedel J, Tanaka M, Tanaka H, Kiyonaga M. et al. Consequences ofone-week creatine supplementation on creatinine levels in athletes'serum and urine, Schweizerische Zeitschrift für <<Sportmedizin andSporttraumatologie>> 2000; 48: 111-116.] Information can also beobtained from the World Wide Web about how to “load” with creatine andprotein, consume large amounts of water prior to the test, and performexercise to obtain both a normal urine creatinine and specific gravitywhile still diluting DOA below detection cut off levels.

Combatting efforts to subvert creatinine measurement start withimproving the accuracy of creatinine assays. Aside from adulteration ofa sample, there are other factors that can interfere with the accuracyof measuring urine creatinine. One of the factors is the presence ofblood cells in urine. DOA such as cocaine, methamphetamines, opioids,benzodiazepines, synthetic marijuana, and others are toxic to the body.The DOA breakdown muscle tissue, including that in the kidneys, whichresults in increased amounts of blood cells in urine. Creatinine assaysof urine samples utilize alkaline picrate to bind to creatinineimparting an orange coloration to the urine. The hemoglobin in bloodcells, also being red, can interfere with the spectrophotometricmeasurement of creatinine in a urine sample.

Embodiments of the subject invention provide a reagent system formeasuring creatinine in a urine sample that reduces the effect of bloodcell hemoglobin when measuring creatinine in the urine. The reagentsystem can include two reagents. The first reagent can include potassiumferricyanide and a detergent, such as, for example, Teepol™. Potassiumferricyanide oxidizes the iron in hemoglobin to a ferric state formingmethemoglobin. This can impart the reacted urine sample with a greencolor, which can be detected and measured spectrophotometrically atbetween about 500 nm and 570 nm, preferably at about 520 nm. This firstspectrophotometric measurement can indicate the amount of hemoglobinthat was in the sample.

In a further embodiment the reacted urine sample is treated with asecond reagent of the reagent system that includes picric acid andsodium hydroxide with a chelating agent, such as, for example,ethylenediaminetetraacetic acid (EDTA). The further reacted sample canbe spectrophotometrically analyzed a second time at between 590 nm and620 nm, preferably about 600 nm. The first spectrophotometricmeasurement of the amount of hemoglobin in the sample can be subtractedfrom the second spectrophotometric measurement to provide a correctedspectrophotometric measure, which can be used to calculate a moreaccurate amount of the creatinine in the sample. Currently usedprotocols mandate that a creatinine value of less than 20 mg/dlindicates the sample is invalid.

Advantageously, embodiments of the reagent system for measuringcreatinine can be used with automated equipment, such as clinicalanalyzers and laboratory equipment typically used tospectrophotometrically analyze a urine sample. One embodiment of aReagent System of the subject invention for measurement of creatininefor use in automated equipment is described below:

Reagent 1: R1 1 Liter To about 800 mL of deionized water add and mix:Tepol (Detergent) 26.0 grams Potassium Ferricyanide 0.094 grams Mix todissolve. Avoid excess exposure to light. Adjust pH to pH 7.5 +/− 0.4with 5% Potassium Hydroxide Bring to 1 Liter total volume with deionizedwater and mix. Store refrigerated @ 2-8 Degrees Centigrade. Reagent 2:R2 1 Liter To about 800 mL of deionized water add and mix: SodiumHydroxide 12.0 grams Disodium EDTA 0.14 grams Picric Acid 2.0 gramsBring to 1 Liter total volume with deionized water and mix. Storerefrigerated at 2°-8° C.

Following are specifications for analyzing urine samples with severaldifferent types of automated equipment, including the Beckman Coulter AU400, AU 400e, AU 480, AU 640, AU640e and AU680 Series Clinical ChemistryAnalyzers. The settings shown are intended to be guidelines for theindicated instruments. It is within the skill of a person trained in theart to recognize that such parameters will vary between instruments.

Assay Parameter Settings Specific Test Parameters 1 Sample Volume 2 μLR1 Volume 25 μL R2 Volume 172 μL Wavelength Primary 520 Secondary 600Method Fixed Reaction Slope + Measuring Point 1 First 13 Last  17Specific Test Parameters 2 L H Normal Range 20 500 Unit mg/dL DecimalPlaces 1 Calibration Specific Calibration Type 3AB Formula POLYNOMIALCounts 2 conc Factor-OD-L Factor-OD-H Point 1 0 −2.0 2.0 Point 2 100−2.0 2.0 Point 3 500 −2.0 2.0V. Assay for Measuring pH of a Urine Sample

Current procedures mandate that urine samples submitted for DOA testingare considered invalid if the pH is below a 3.0 cut-off value or above a10.0 cut-off value. Automated pH measurement using a mixture of pHindicator dyes has been used for several years in DOA testing. Thereagents use a dye mixture producing a single color. However, thesecommonly used reagents suffer from limited ranges of detection andimprecision in the low pH range. Both of these may be attributed to thefact that the color development increases progressively from acid toalkaline pH levels. Thus, there are very low absorbance values at verylow acid pH levels. Both the limited range of detection and the lowsensitivity in the acid range limit the optimal detection of the commonacid or alkali addition subversion means. A better range of measurementand more precise measurement in the acid range are required for optimaluse in the diagnosis of SUD.

An improved, automated pH screening reagent and method have beendeveloped for use as components of the SUD detection panel. The newreagent advantageously includes pH indicator dyes that form differentcolors in the acid and alkaline pH extremes. Colorimetric detection canbe made using the bi-chromatic feature available on automated systems.The reagent uses indicator dyes that form a red color in the acid pHrange (absorbance wavelength 510-520 nm) and other indicator dyes thatform blue colors in the alkaline range (absorbance wavelength 590-610nm). The 600 nm wavelength is used as a primary detection wavelength andthe 510-520 wavelength is used as the secondary bi-chromatic wavelength.FIG. 11 shows the pH ranges and associated colors for the pH indicatordyes utilized with embodiments of the pH assay of the subject invention.

Bi-chromatic detection has been used on automated systems to correct forturbidity interference. The primary wavelength is used to detect thecolor formed by the reaction and the higher secondary wavelength is usedto detect the absorbance of turbidity. The absorbance at the secondarywavelength is subtracted from the absorbance at the primary wavelengthto correct for turbidity (Net Absorbance is Netabsorbance=(Abs_(Primary)−Abs_(secondary)). The bi-chromatic turbiditycorrection is a standard feature on virtually all clinical chemistryanalyzers is use today. Embodiments of the pH screening reagent andmethod of the subject invention uses the bi-chromatic turbiditycorrection arithmetic of the analyzers in a different method. Ratherthan correcting for turbidity, the effect of two different colors ofdyes on a urine sample is measured. The extreme acid pH indicating dyesproduce a highly negative net absorbance as the red colored dyes absorbat the secondary wavelength (510-520 nm) and no blue color is present,so that absorption at the primary wavelength is very low. At alkaline pHextremes, the color is blue and there is no red color present. Thusthere is high absorbance at the primary wavelength and very little atthe secondary wavelength thereby producing a high positive netabsorbance. This is illustrated in the graph in FIG. 12.

The formulation for 1 liter of the reagent is as follows:

STEP A: To 60 mL of methanol containing 3 drops of 1 N NaOH add:

-   -   0.015 grams of Thymolphthalein    -   0.003 grams Xylenol Blue water soluble    -   0.005 grams Bromothymol Blue and Mix all ingredients to        dissolve.

STEP B: To 700 mL deionized water add:

-   -   0.5 grams ProClin™ 300    -   0.004 grams Methyl Orange and mix to dissolve.    -   0.028 grams of Litmus

STEP C:

-   -   Add mixture from Step A and adjust pH to 7.4+/−0.1 with dilute        sodium hydroxide or hydrochloric acid. Then bring to volume of 1        Liter with deionized water.

Calibrators having a buffered pH of 3.0 and 10.5 prepared by known meansare used.

Following are specifications for analyzing urine samples utilizingBeckman Coulter automated equipment, including the AU 400, AU 400e, AU480, AU 640, AU640e and AU680 Series Clinical Chemistry Analyzers. Thesettings shown are intended to be guidelines for the indicatedinstruments. It is within the skill of a person trained in the art torecognize that such parameters will vary between instruments.

Sample Volume  25 R1 Volume 150 Wavelength Primary 600 WavelengthSecondary 520 Measuring Point 1 First 0 Last 17 Unit: pH Decimal Places:1 Counts: 2 Process: OD Calibration Type A Formula Y= AX + B Point 1CONC:   3.0 Point 2 CONC  10.5

Substance Abuse and Mental Health Services Administration (SAMHSA)currently defines two principle means of detecting SUD: (a) interviewand observation of behavior, and (b) by detection of efforts atsubversion to hide or mask use of drugs-of-abuse (DOA). Of the twoprinciple means, only the second one provides an objective result. Thereare twelve recognized subversion techniques. The embodiments of thesubject invention provide a SUD Diagnostic Panel that includes sixassays capable of detecting all twelve subversion techniques. Thisallows for more accurate validation of sample integrity beforeconducting further tests of DOA. It also provides an objective diagnosisof SUD.

The scope of the invention is not limited by the specific examples andsuggested procedures and uses related herein since modifications can bemade within such scope from the information provided by thisspecification to those skilled in the art.

All patents, patent applications, provisional applications, and otherpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

What is claimed is:
 1. A method for diagnosing Substance Use Disorder(SUD), adapted for use with a urine sample, that comprises, analyzingthe urine sample spectrophotometrically with a SUD Diagnostic Panelcomprising: an Oxidant History assay that utilizes a phosphotungstatereagent to measure uric acid in the sample and compare the amount ofuric acid to a pre-determined value, such that an amount of uric acidbelow the pre-determined value indicates the urine sample is invalid; ashort-duration counterfeit urine assay that utilizes thymolphthaleinmonophosphate to detect the presence of an acid phosphatase marker inthe urine sample, such that failure to detect the acid phosphatasemarker indicates the sample is invalid; a long-duration counterfeiturine assay that utilizes p-nitrophenyl phosphate to detect the presenceof an alkaline phosphatase marker in the urine sample, such that failureto detect the alkaline phosphatase marker indicates the sample isinvalid; a Specific Gravity Index assay that utilizes β-Galactosidaseand o-nitrophenylgalactoside to measure a combined amount of sodium andpotassium in the urine sample to obtain a Specific Gravity Index value,such that a Specific Gravity Index value that is outside of apre-determined range indicates the sample is invalid; a creatinine assaythat utilizes alkaline picrate to measure the amount of creatinine inthe urine sample, such that an amount of creatinine outside of apre-determined range indicates the sample is invalid; and a pH assaythat bi-chromatically measures pH of the urine sample, such that a pH inthe acid range presents a different color than a pH in the alkalinerange; determining if at least one of the SUD Diagnostic Panel assaysindicate the urine sample is invalid, thereby providing an objectiveindication of SUD.
 2. The method according to claim 1, wherein theOxidant History reagent comprises a phosphotungstate.
 3. The method,according to claim 2, wherein the Oxidant History assay determines ahistorical presence of an oxidative-adulterant in a urine sample, by:reacting the urine sample with a reagent comprising phosphotungstate;analyzing the reacted urine sample using spectrophotometry to obtain afirst light absorbance measurement of the reacted urine sample;calculating a first Uric Acid Equivalents value utilizing the firstlight absorbance measurement; analyzing the reacted urine sample againby spectrophotometry, after a pre-determined amount of time; obtainingan additional light absorbance measurement of the reacted urine sample;calculating an additional Uric Acid Equivalents value utilizing theadditional light absorbance measurement; utilizing the first Uric AcidEquivalents value and the additional Uric Acid Equivalents value toobtain an Oxidant History for the urine sample; and analyzing theOxidant History to determine whether the first Uric Acid Equivalentvalue is larger than the additional Uric Acid Equivalent value, whichindicates the historical presence of an oxidative-adulterant in theurine sample.
 4. The method, according to claim 3, wherein analysis ofthe reacted sample is configured to be performed at a wavelength in arange between 580 nm and 800 nm.
 5. The method according to claim 4,wherein any Uric Acid Equivalents value below 10 mg/dl is indicative ofthe urine sample being invalid.
 6. The method according to claim 1,wherein the p-nitrophenyl phosphate of the long-duration counterfeiturine assay catalyzes to p-nitrophenol.
 7. The method according to claim1, wherein the thymolphthalein monophosphate of the short-durationcounterfeit urine assay catalyzes to thymolphthalein.
 8. The methodaccording to claim 1, further comprising conducting analysis of theurine sample with the SUD Diagnostic Panel prior to conducting analysisfor drugs of abuse.
 9. The method according to claim 8, furthercomprising conducting analysis of a retaken urine sample with the SUDDiagnostic Panel prior to conducting analysis for drugs of abuse. 10.The method according to claim 9, wherein a determination that theretaken sample is invalid is an objective diagnosis of SUD.
 11. Themethod according to claim 9, wherein a valid sample is analyzed fordrugs of abuse utilizing liquid or gas chromatography/mass spectrometryprocedures.
 12. The method according to claim 11, wherein detection ofdrugs of abuse in the retaken sample is an objective diagnosis of SUD.13. A method for measuring the amount of creatinine in a urine sample,utilizing a reagent system according to claim 8, comprising: reacting aurine sample with the first reagent, such that hemoglobin in the sampleis converted to methemoglobin, obtaining a first spectrophotometricmeasurement of the sample, reacting the urine sample further with thesecond reagent; obtaining a second spectrophotometric measurement of thefurther reacted sample; subtracting the first spectrophotometricmeasurement from the second spectrophotometric measurement to obtain acorrected spectrophotometric measurement; calculating an amount ofcreatinine in the sample utilizing the corrected spectrophotometricmeasurement.
 14. The method according to claim 13, further comprisingspectrophotometrically analyzing the reacted urine sample at between 500nm and 570 nm.
 15. The method according to claim 14, further comprisingspectrophotometrically analyzing the reacted urine sample at about 520nm.
 16. The method according to claim 13, further comprisingspectrophotometrically analyzing the further reacted urine sample atbetween 590 nm and 620 nm.
 17. The method according to claim 16, furthercomprising spectrophotometrically analyzing the further reacted urinesample at about 600 nm.